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Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water

  • Applied genetics and molecular biotechnology
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Abstract

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliC h7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.

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References

  • Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell, 4th edn. Garland Science, New York

    Google Scholar 

  • Albuquerque P, Mendes MV, Santos CL, Moradas-Ferreira P, Tavares F (2009) DNA signature-based approaches for bacterial detection and identification. Sci Total Environ 407:3641–3651

    Article  CAS  PubMed  Google Scholar 

  • Atlas RM (2004) Handbook of microbiological media, 3rd edn. CRC Press.

  • Bhagwat AA (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int J Food Microbiol 84:217–224

  • Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, Peters G, Karch H (2011) Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis 11:671–676

    Article  CAS  PubMed  Google Scholar 

  • Bischoff C, Luthy J, Altwegg M, Baggi F (2005) Rapid detection of diarrheagenic E. coli by real-time PCR. J Microbiol Methods 61:335–341

    Article  CAS  PubMed  Google Scholar 

  • Bloch S, Felczykowska A, nejman-Falen’czyk B (2012) Escherichia coli O104:H4 outbreak—have we learnt a lesson from it? Acta Biochim Pol 59:483–488

    CAS  PubMed  Google Scholar 

  • Bonnet R, Souweine B, Gauthier G, Rich C, Livrelli V, Sirot J, Joly B, Forestier C (1998) Non-O157:H7 Stx2-producing Escherichia coli strains associated with sporadic cases of hemolytic-uremic syndrome in adults. J Clin Microbiol 36:1777–1780

    CAS  PubMed Central  PubMed  Google Scholar 

  • CDC (2001) University outbreak of calicivirus infection mistakenly attributed to Shiga toxin-producing Escherichia coli O157: H7– Virginia, 2000. MMWR Morb Mortal Wkly Rep 50:489–491

    Google Scholar 

  • CDC (2006) Importance of culture confirmation of Shiga toxin-producing Escherichia coli infection as illustrated by outbreaks of gastroenteritis—New York and North Carolina, 2005. MMWR Morb Mortal Wkly Rep 55:1042–1045

    Google Scholar 

  • Cebula TA, Payne WL, Feng P (1995) Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol 33:248–250

    CAS  PubMed Central  PubMed  Google Scholar 

  • Chen J, Zhang L, Paoli GC, Shi C, Tu SI, Shi X (2010) A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis. Int J Food Microbiol 137:168–174

    Article  CAS  PubMed  Google Scholar 

  • Cho MS, Kang MJ, Kim C, Seol Y, Hahn JH, Park SC, Hwang DJ, Ahn T, Park DH, Lim CK, Park DS (2011) Sensitive and specific detection of Xanthomonas oryzae pv. oryzae by real-time bio-PCR using pathovar-specific primers based on an rhs family gene. Plant Dis 95:589–594

    Article  CAS  Google Scholar 

  • Feng P, Lampel KA (1994) Genetic analysis of uidA expression in enterohaemorrhagic Escherichia coli serotype O157:H7. Microbiology 140:2101–2107

    Article  CAS  PubMed  Google Scholar 

  • Feng PC, Jinneman K, Scheutz F, Monday SR (2011) Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes. Appl Environ Microbiol 77:6699–6702

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jothikumar N, Griffiths MW (2002) Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Appl Environ Microbiol 68:3169–3179

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kim HJ, Park SH, Lee TH, Nahm BH, Chung YH, Seo KH, Kim HY (2006) Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars. J Food Prot 69:1653–1661

    CAS  PubMed  Google Scholar 

  • Lang JM, Hamilton JP, Diaz MGQ, Van Sluys MA, Burgos MRG, Vera Cruz CM, Buell CR, Tisserat NA, Leach JE (2010) Genomics-based diagnostic marker development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Dis 94:311–319

    Article  CAS  Google Scholar 

  • Lin CK, Lin JC (2007) Development of PCR primers based on a fragment from randomly amplified polymorphic DNA for the detection of Escherichia coli O157:H7/NM. Mol Cell Probes 21:182–189

    Article  CAS  PubMed  Google Scholar 

  • Mukhopadhyay UM, Mukhopadhyay A (2007) Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli O157:H7 and Listeria monocytogenes. J Microbiol Meth 68:193–200

    Article  CAS  Google Scholar 

  • Müller D, Greune L, Heusipp G, Karch H, Fruth A, Tschäpe H, Schmidt MA (2007) Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR. Appl Environ Microbiol 73:3380–3390

    Article  PubMed Central  PubMed  Google Scholar 

  • Muniesa M, Blanco JE, De Simón M, Serra-Moreno R, Blanch AR, Jofre J (2004) Diversity of stx2 converting bacteriophages induced from Shiga-toxin-producing Escherichia coli strains isolated from cattle. Microbiology 150:2959–2971

    Article  CAS  PubMed  Google Scholar 

  • Narang N, Fratamico PM, Tillman G, Pupedis K, Cray WC Jr (2009) Performance comparison of a fliC h7 real-time PCR assay with an H7 latex agglutination test for confirmation of the H type of Escherichia coli O157:H7. J Food Prot 72:2195–2197

    CAS  PubMed  Google Scholar 

  • Nielsen EM, Andersen MT (2003) Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5′ nuclease PCR assay. J Clin Microbiol 41:2884–2893

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Paton AW, Paton JC, Goldwater PN, Manning PA (1993) Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures using the polymerase chain reaction. J Clin Microbiol 31:3063–3067

    CAS  PubMed Central  PubMed  Google Scholar 

  • Paton AW, Paton JC (1998) Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J Clin Microbiol 36:598–602

    CAS  PubMed Central  PubMed  Google Scholar 

  • Paton AW, Paton JC (2002) Direct detection and characterization of shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa. J Clin Microbiol 40:271–274

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Perelle S, Dilasser F, Grout J, Fach P (2004) Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world’s most frequent clinical cases. Mol Cell Probes 18:185–192

    Article  CAS  PubMed  Google Scholar 

  • Segerman B, De Medici D, Ehling Schulz M, Fach P, Fenicia L, Fricker M, Wielinga P, Van Rotterdam B, Knutsson R (2011) Bioinformatic tools for using whole genome sequencing as a rapid high resolution diagnostic typing tool when tracing bioterror organisms in the food and feed chain. Int J Food Microbiol 145:S167–S176

    Article  CAS  PubMed  Google Scholar 

  • Tiba MR, Cd M, Carazzolle MF, Leite Dda S (2011) Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFLP and DNA sequencing methods. Braz J Infect Dis 15:144–150

    Article  CAS  PubMed  Google Scholar 

  • Whelan JA, Russel NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real time PCR. J Immunol Methods 278:261–269

    Article  CAS  PubMed  Google Scholar 

  • Yuan Y, Xu W, Zhai Z, Shi H, Luo Y, Chen Z, Huang K (2009) Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples. J Food Sci 74:446–452

    Article  Google Scholar 

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Acknowledgments

This study was performed with the support of “Research Program for Agricultural Science & Technology Development (Project no. PJ008612),” the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. National Academy of Agricultural Science, Rural Development Administration, Suwon, 441-707, Republic of Korea

    Min Seok Cho & Dong Suk Park

  2. Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin, Gyeonggi, 449-791, Republic of Korea

    Kiseong Joh

  3. Department of Microbiology, Dankook University, Cheonan, 330-714, Republic of Korea

    Min Seok Cho & Tae-Young Ahn

Authors
  1. Min Seok Cho
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  2. Kiseong Joh
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  3. Tae-Young Ahn
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  4. Dong Suk Park
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Correspondence to Dong Suk Park.

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Cho, M.S., Joh, K., Ahn, TY. et al. Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water. Appl Microbiol Biotechnol 98, 7869–7877 (2014). https://doi.org/10.1007/s00253-014-5855-8

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  • DOI: https://doi.org/10.1007/s00253-014-5855-8

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